The chromatographic separation, which enables separation of a desired component from a solution containing a plurality of dissolved components in a simple manner with less energy, has come to be utilized industrially in the fields of separation and purification of medicines, foods, and the like.
For the chromatographic separation apparatuses employed therefor, two types of packed columns are known: namely, a first type of packed columns in which a separating agent is packed throughout the entire column, and a second type of packed columns in which a separating agent is packed partially in the column so as to keep a space in the top portion of the column for allowing back-wash of the separating agent (the latter type being referred to hereinafter as "top-spaced type").
In chromatographic separation-purification, the unseparated liquid is pretreated for eliminating suspended matters by filtration or the like procedure, and is then passed through the packed column. After the liquid has been passed for a long period of time, fine suspended matters accumulate in the packed layer in the column, causing pressure drop in the packed column and lowering liquid flow rate, whereby the liquid cannot be passed through under a constant pressure.
Accordingly, the top-spaced type of packed columns, which permit the back-wash for discharging the suspended matter from the packed column, is widely employed in industrial chromatographic separation apparatuses.
In this top-spaced type, generally an unseparated liquid or an eluting liquid is passed through the column by holding and retaining a layer of the respective liquid above the packed layer to prevent any deflection of flow in the packed layer. For high precision chromatographic separation, the thickness of the retained liquid layer should be kept constant within a variable range of from 10 to 20 cm at most.
However, in the case where a separating agent employed is of a polymer type such as an ion-exchange resin, a synthetic organic adsorbent, or the like, the separating agent swells or shrinks depending on the pH, the salt concentration, the liquid properties of the passing liquid, and other factors.
Such a phenomenum, for example, is observed that a strongly acidic cation-exchange resin employed as a packing agent will shrink when an electrolyte solution such as aqueous sodium chloride solution is passed as an elution liquid after passing of the unseparated liquid, and will swell and restore the original volume when the unseparated liquid is passed again.
Accordingly, in a top-spaced type packed column having polymeric separating agent packed therein, not only the thickness of the packed layer but also the packing density of the packed layer incessantly changes gradually, whereby the thickness of the retained liquid layer changes correspondingly.
Such variation of the layer thickness of the retained liquid layer naturally leads to lowering of the separating performance. Therefore, the variation of the layer thickness of the retained liquid needs to be controlled to be smallest during the passing-through of the liquid. The control, however, cannot be successful without extremely troublesome operations.
The chromatographic separation by a conventional top-spaced type of packed column, therefore, is compelled to be conducted with the layer thickness of the retained liquid varying in a considerably broad range. This variation of the layer thickness is accompanied naturally with dilution of the unseparated liquid or the elution liquid by the elution liquid or the unsaturated liquid having been employed in the preceding step, which involves such disadvantage that the purity of the intended substance cannot be raised as desired.